Our long-term objectives are to understand the evolution of arboviruses in the context of their transmission cycles and how this impacts viral emergence, adaptation and persistence. WNV genetic diversity is greater in mosquitoes than birds in both nature and the laboratory. The exact reasons for this difference are unknown, but may be the result of divergent host antiviral immune responses. Vertebrates respond to viral infections mainly via type-1 interferon and natural killer cells, whereas mosquitoes rely on RNA interference (RNAi) as an intracellular defense mechanism to control viral infections. The sequence-specificity of RNAi may drive increases in genetic diversity through negative frequency-dependent selection within host cells, and consequently within hosts. Therefore, we propose a series of in vitro and in vivo experiments to elucidate the contribution of the RNAi-based antiviral response on WNV genetic diversification. In Aim 1 we will determine if induction and/or suppression of the RNAi pathway in mosquito and chicken cells results in gains and reductions in WNV genetic diversity, respectively. Likewise, Aim 2 will determine if induction and/or suppression of the RNAi pathway in Cx. pipiens results in gains or reductions in WNV genetic diversity, respectively. Using short-hairpin RNAs in Aim 1 we will either induce (WNV-specific ShRNAs) or suppress (dcr-2 and ago-2-specific shRNAs) the RNAi response in mosquito and chicken cell culture and assess the impacts on WNV genetic diversity. Similarly, long, double-stranded RNA molecules either targeting the WNV genome or dcr-2 and ago-2 will be used to test for similar outcomes in adult female Cx. pipiens. For both Aims suppression will be monitored by Q-RT-PCR and viral population diversity analyzed by high-throughput sequencing. The overall hypothesis is that induction of the RNAi pathway with either WNV-specific shRNAs or dsRNAs will result in genetic gains in mosquito and chicken cells and adult mosquitoes. Alternatively, suppression of the RNAi response with dcr-2 and ago-2-specific shRNAs or dsRNAs will result in decreases in genetic diversity in adult mosquitoes and mosquito cell culture, but not in chicken cell culture. PUBLIC HEALTH RELEVANCE: The introduction and adaptation of arboviruses to new ecological niches pose an ongoing threat to human health. The recent epidemics of West Nile virus in North America and chikungunya virus (CHIKV) in the Eastern hemisphere highlight the potential for other arbovirus such as dengue virus (DENV) and Rift Valley fever virus (RVFV) to emerge in non-endemic regions. Understanding the adaptive mechanisms of arboviruses is paramount to developing and implementing effective control measures.